Irreversible Interaction Research Articles

The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1

The polo kinase family are important oncology targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compounds containing the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compounds that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-containing molecules in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compounds that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.

Three-dimensional microfluidic chip with twin-layer herringbone structure for high efficient tumor cell capture and release via antibody-conjugated magnetic microbeads.

Harvesting rare circulating tumor cells (CTCs) from human blood is distinctly substantial to monitor tumor stage and evaluate therapeutic efficacy. As a proof-of-concept study, a microfluidic chip with twin-layer herringbone grooves was developed to isolate and recover tumor cells with high efficiency based on the immunoreaction between cells and antibody-conjugated microbeads (MBs) under local magnetic field. Functional MBs were initially localized onto the internal channel wall through the magnetic guidance. Then, infused tumor cells were deviated into the herringbone groove via passive microvortex and were further trapped through an irreversible interaction with MBs. Upon the removal of magnet, the captured cells and residual MBs were released from the channel and collected for further analysis in cell adhesion and proliferation in vitro. Capture efficiency of tumor cells reached up to ∼90% and limit of detection was down to 50 cells per mL based on this approach. Furthermore, recovery rate of tumor cells was as high as ∼94%, and potencies of cell attachment and proliferation was well maintained in retrieved cells. Hence, the present technique has a great potential for the isolation, quantitation and recovery of CTCs for cancer theranostic guidance and biomolecular analysis.

Manipulation of entanglement sudden death in an all-optical setup

The unavoidable and irreversible interaction between an entangled quantum system and its environment causes decoherence of the individual qubits as well as degradation of the entanglement between them. Entanglement sudden death (ESD) is the phenomenon wherein disentanglement happens in finite time even when individual qubits decohere only asymptotically in time due to noise. Prolonging the entanglement is essential for the practical realization of entanglement-based quantum information and computation protocols. For this purpose, the local NOT operation in the computational basis on one or both qubits has been proposed. Here, we formulate an all-optical experimental set-up involving such NOT operations that can hasten, delay, or completely avert ESD, all depending on when it is applied during the process of decoherence. Analytical expressions for these are derived in terms of parameters of the initial state's density matrix, whether for pure or mixed entangled states. After a discussion of the schematics of the experiment, the problem is theoretically analyzed, and simulation results of such manipulations of ESD are presented.

Novel Irreversible Agonists Acting at the A1 Adenosine Receptor.

The A1 adenosine receptor (A1AR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the A1AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesized, and four compounds, 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the A1AR. Of these compounds, 15b and 15d were more directly validated as irreversible agonists of the A1AR using membrane-based [3H]DPCPX and [35S]GTPγS binding experiments.

Ibrutinib Inhibits ERBB Receptor Tyrosine Kinases and HER2-Amplified Breast Cancer Cell Growth.

Ibrutinib is a potent, small-molecule Bruton tyrosine kinase (BTK) inhibitor developed for the treatment of B-cell malignancies. Ibrutinib covalently binds to Cys481 in the ATP-binding domain of BTK. This cysteine residue is conserved among 9 other tyrosine kinases, including HER2 and EGFR, which can be targeted. Screening large panels of cell lines demonstrated that ibrutinib was growth inhibitory against some solid tumor cells, including those inhibited by other HER2/EGFR inhibitors. Among sensitive cell lines, breast cancer lines with HER2 overexpression were most potently inhibited by ibrutinib (<100 nmol/L); in addition, the IC50s were lower than that of lapatinib and dacomitinib. Inhibition of cell growth by ibrutinib coincided with downregulation of phosphorylation on HER2 and EGFR and their downstream targets, AKT and ERK. Irreversible inhibition of HER2 and EGFR in breast cancer cells was established after 30-minute incubation above 100 nmol/L or following 2-hour incubation at lower concentrations. Furthermore, ibrutinib inhibited recombinant HER2 and EGFR activity that was resistant to dialysis and rapid dilution, suggesting an irreversible interaction. The dual activity toward TEC family (BTK and ITK) and ERBB family kinases was unique to ibrutinib, as ERBB inhibitors do not inhibit or covalently bind BTK or ITK. Xenograft studies with HER2+ MDA-MB-453 and BT-474 cells in mice in conjunction with determination of pharmacokinetics demonstrated significant exposure-dependent inhibition of growth and key signaling molecules at levels that are clinically achievable. Ibrutinib's unique dual spectrum of activity against both TEC family and ERBB kinases suggests broader applications of ibrutinib in oncology. Mol Cancer Ther; 15(12); 2835-44. ©2016 AACR.

Improvement of catalytic performance of lignin peroxidase for the enhanced degradation of lignocellulose biomass based on the imbedded electron-relay in long-range electron transfer route.

BackgroundAlthough lignin peroxidase is claimed as a key enzyme in enzyme-catalyzed lignin degradation, in vitro enzymatic degradation of lignin was not easily observed in lab-scale experiments. It implies that other factors may hinder the enzymatic degradation of lignin. Irreversible interaction between phenolic compound and lignin peroxidase was hypothesized when active enzyme could not be recovered after the reaction with degradation product (guaiacol) of lignin phenolic dimer.ResultsIn the study of lignin peroxidase isozyme H8 from white-rot fungi Phanerochaete chrysosporium (LiPH8), W251 site was revealed to make the covalent coupling with one moiety of monolignolic radical (guaiacol radical) by LC-MS/MS analysis. Hypothetical electron-relay containing W251 residue was newly suggested based on the observation of repressed radical coupling and remarkably lower electron transfer rate for W215A mutant. Furthermore, the retardation of the suicidal radical coupling between the W251 residue and the monolignolic radical was attempted by supplementing the acidic microenvironment around the W251 residue to engineer radical-robust LiPH8. Among many mutants, mutant A242D showed exceptional catalytic performances by yielding 21.1- and 4.9-fold higher increases of kcat and kcat/KM values, respectively, in the oxidation of non-phenolic model lignin dimer.ConclusionsA mechanism-based suicide inhibition of LiPH8 by phenolic compounds was firstly revealed and investigated in this work. Radical-robust LiPH8 was also successfully engineered by manipulating the transient radical state of radical-susceptible electron-relay. Radical-robust LiPH8 will play an essential role in degradation of lignin, which will be consequently linked with improved production of sugars from lignocellulose biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0664-1) contains supplementary material, which is available to authorized users.

Kynurenine Aminotransferase Isozyme Inhibitors: A Review.

Kynurenine aminotransferase isozymes (KATs 1–4) are members of the pyridoxal-5’-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies.

Interactions between Silicon Oxide Nanoparticles (SONPs) and U(VI) Contaminations: Effects of pH, Temperature and Natural Organic Matters.

The interactions between contaminations of U(VI) and silicon oxide nanoparticles (SONPs), both of which have been widely used in modern industry and induced serious environmental challenge due to their high mobility, bioavailability, and toxicity, were studied under different environmental conditions such as pH, temperature, and natural organic matters (NOMs) by using both batch and spectroscopic approaches. The results showed that the accumulation process, i.e., sorption, of U(VI) on SONPs was strongly dependent on pH and ionic strength, demonstrating that possible outer- and/or inner-sphere complexes were controlling the sorption process of U(VI) on SONPs in the observed pH range. Humic acid (HA), one dominated component of NOMs, bounded SONPs can enhance U(VI) sorption below pH~4.5, whereas restrain at high pH range. The reversible sorption of U(VI) on SONPs possibly indicated that the outer-sphere complexes were prevalent at pH 5. However, an irreversible interaction of U(VI) was observed in the presence of HA (Fig 1). It was mainly due to the ternary SONPs-HA-U(VI) complexes (Type A Complexes). After SONPs adsorbed U(VI), the particle size in suspension was apparently increased from ~240 nm to ~350 nm. These results showed that toxicity of both SONPs and U(VI) will decrease to some extent after the interaction in the environment. These findings are key for providing useful information on the possible mutual interactions among different contaminants in the environment.

A Bisbenzamidine Phosphonate as a Janus-faced Inhibitor for Trypsin-like Serine Proteases.

A hybrid approach was applied for the design of an inhibitor of trypsin-like serine proteases. Compound 16 [(R,R)- and (R,S)-diphenyl (4-(1-(4-amidinobenzylamino)-1-oxo-3-phenylpropan-2-ylcarbamoyl)phenylamino)(4-amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus-faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second-order rate constant (kinac /Ki) of 59 500 M(-1) s(-1). With human matriptase, 16 showed preference for a reversible mode of inhibition (IC50 =2.6 μM) as indicated by linear progress curves and enzyme reactivation.

Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus.

BackgroundIn adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus.MethodsWe constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution.ResultsLike in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed.ConclusionsChimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.

Ca2+ -myristoyl switch in neuronal calcium sensor-1: a role of C-terminal segment.

NCS1 (neuronal calcium sensor-1) is a Ca(2+)-myristoyl switch protein of the NCS protein family involved in synaptic plasticity and neurotransmission via Ca(2+)-dependent regulation of dopamine D2 receptor and associated Gprotein coupled receptor kinase (GRK)-2. Overexpression of NCS1 in synaptic terminals results in accumulation of membrane-bound protein and its redundant regulatory activity associated with neurological disorders. Here, we have demonstrated that bovine photoreceptors contain NCS1 that is capable of a partially irreversible interaction with isolated photoreceptor membranes and implicated in Ca(2+)-dependent binding and regulation of GRK1 in vitro. Using NCS1- recoverin C-terminal chimeric construct (NR), it was found that the Ca(2+)-myristoyl switch of NCS1 is affected by its C-terminal segment downstream the fourth EF-loop of the protein, which is variable within the NCS family. NR retains structural stability and sensitivity to Ca(2+), but interacts with photoreceptor membranes with lower affinity in a Ca(2+)- dependent fully reversible manner and displays altered GRK1 modulation. These data combined with fluorescent probing of surface hydrophobicity of NCS1, NR and recoverin suggest that the C-terminal segment of NCS1 regulates reuptake of myristoyl group under Ca(2+)-free conditions and participates in organization of the target-binding pocket of the protein. We point out a putative role of NCS1 in photoreceptors as a modulator of GRK activity and propose targeting of the C-terminal segment of NCS1 as an appropriate way for selective suppression of excessive membrane accumulation and aberrant activity of the protein in neurons associated with central nervous system dysfunctions.

Protein release from water-swellable poly(d,l-lactide-PEG)-b-poly(ϵ-caprolactone) implants

In this study, water-swellable multiblock copolymers composed of semi-crystalline poly(ϵ-caprolactone) [PCL] blocks and amorphous blocks consisting of poly(d,l-lactide) (PDLLA) and poly(ethylene glycol) (PEG) [PDLLA–PEG] were synthesized. The block ratio of these [PDLLA–PEG]-b-[PCL] multiblock copolymers was varied and the degradation of implants prepared of these polymers by hot melt extrusion (HME) was compared with implants prepared of [PCL–PEG]-b-[PCL], a copolymer which has been described previously (Stanković et al., 2014). It was shown that the initial degradation rate of the [PDLLA–PEG]-b-[PCL] multiblock copolymers increased with increasing the content of amorphous [PDLLA–PEG] block and that the degradation rate of these multiblock copolymers was faster than that of the [PCL–PEG]-b-[PCL] multiblock copolymers due to rapid degradation of the [PDLLA–PEG] block.Furthermore, the release of the model proteins lysozyme and bovine serum albumin from polymer implants prepared by HME was studied. It was found that the protein release from [PDLLA–PEG]-b-[PCL] copolymers was incomplete, which is not acceptable for any application of these polymers. Besides, [PCL–PEG]-b-[PCL] copolymers showed slow and continuous release. We hypothesize that the incomplete release is explained by an irreversible interaction between the proteins and polymer degradation products or by entrapment of the protein in the hydrophobic and non-swellable polymer matrix that was left after degradation and loss of the hydrophilic [PDLLA–PEG] blocks from the degrading polymer.

Submicrometer‐Sized ZIF‐71 Filled Organophilic Membranes for Improved Bioethanol Recovery: Mechanistic Insights by Monte Carlo Simulation and FTIR Spectroscopy

Template‐free self‐assembly synthesis of nano‐sized metal‐organic frameworks (MOFs) is of particular interest in MOF research since organized nanostructures possessing distinctive properties are useful for many advanced applications. In this work, the facile room temperature synthesis of robust submicrometer‐sized ZIF‐71 crystals with different particle sizes (140, 290, or 430 nm), having a high permanent microporosity (SBET = 827 cm2 g−1) and synthesis yield up to 80% based on Zn on a gram‐scale, is reported. These small ZIF‐71 particles are ideal filler for the fabrication of thinner and homogeneous polydimethylsiloxane (PDMS) based mixed matrix membranes (MMMs) with excellent filler dispersion and filler‐polymer adhesion at high loading up to 40 wt%, as confirmed by scanning electron microscopy. Pervaporation tests using these submicrometer‐sized ZIF‐71 filled MMMs show significant improvement for bioethanol recovery. Interesting phenomena of i) reversible ethanol‐ethanol hydrogen interaction in the ethanol liquid‐phase and ii) irreversible hydrogen interaction of ethanol and –Cl functional group in the α‐cages and octagonal prismatic cages of ZIF‐71 in ethanol vapor‐phase are discovered for the first time by a Fourier transform infrared spectroscopy (FTIR) study. In full agreement with molecular simulation results, these explain fundamentally the ZIF‐71 filled MMMs pervaporation performance.

삼화 원탄과 무회분탄의 촉매(K2CO3) 가스화 반응성 비교 연구

석탄의 가스화는 촉매 도입 시 온순 조건에서 가능하나, 석탄 내 회분에 의한 비활성화에 의해 반복적인 촉매 활용이 힘들다. 이에 본 연구에서는 삼화 원탄에서 회분을 제거하여 삼화 무회분탄(ash-free coal, AFC)을 제조한 후 가스화 반응성을 원탄과 비교하여 알아보았다. 우선 원탄을 대상으로 고정층 반응기에서 수증기 공급량, 공간 속도(space velocity), 온도 및 촉매를 변수로서 가스화 조건을 결정하였다. 고체상 혼합법으로 다양한 촉매 도입 시, 유동성을 갖는 <TEX>$K_2CO_3$</TEX>가 가장 높은 활성을 보였다. 무회분탄은 원탄보다 낮은 반응성을 보였으며, 이는 용매(1-methylnaphthalene, 1-MN)를 이용한 고온 추출 및 건조 공정 중에 소모된 산소 기능기 함량과 증가된 탄화도(carbonization)에 기인한다. <TEX>$K_2CO_3$</TEX> 가 혼합된 무회분탄의 반응성은 급격히 증가하여 낮은 온도 (<TEX>$700^{\circ}C$</TEX>)에서도 높은 전환율을 보였다. 이때 <TEX>$H_2/CO$</TEX>와 <TEX>$CO_2/CO$</TEX> 비율도 증가하는데, 이는 촉매에 의해 수성가스전환(water-gas shift) 반응이 활성화됨에 기인한다. 본 연구에서는 무회분탄의 저온 촉매 가스화 반응을 통해 석탄 가스화 공정의 경제성이 개선될 수 있음을 확인하였다. Catalytic gasification of raw coals at mild condition is not realized yet mainly due to deactivation of catalysts via their irreversible interaction with mineral matters in coal. In this work, the gasification behavior of ash-free coal (AFC) was compared with that of the parent raw coal. In order to modify the gasification conditions, the raw coal gasified with fixed variables (water supply, space velocity, temperature, catalysts) in a fixed bed reactor. When catalysts are added by physical mixing method with coal, <TEX>$K_2CO_3$</TEX> was the most effective additives for steam gasification of coal. However, the activity of ash-free coal (AFC) was much less reactive than raw coal due to high temperature extraction in a 1-methylnaphthalene under 30bar at <TEX>$370^{\circ}C$</TEX> for 1 h, almost removed oxygen functional groups, and increased carbonization. The addition of <TEX>$K_2CO_3$</TEX> in AFC achieved higher conversion rate at low temperature (<TEX>$700^{\circ}C$</TEX>). At that time, the molar ratio of gases (<TEX>$H_2/CO$</TEX> and <TEX>$CO_2/CO$</TEX>) was increased because of water-gas shift reaction (WGSR) by addition of catalysts. This shows that catalytic steam gasification of AFCs is achievable for economic improvement of gasification process at mild temperature.

SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.

The capture of biotin by streptavidin is an inspiration for supramolecular chemistry and a central tool for biological chemistry and nanotechnology, because of the rapid and exceptionally stable interaction. However, there is no robust orthogonal interaction to this hub, limiting the size and complexity of molecular assemblies that can be created. Here we combined traptavidin (a streptavidin variant maximizing biotin binding strength) with an orthogonal irreversible interaction. SpyTag is a peptide engineered to form a spontaneous isopeptide bond to its protein partner SpyCatcher. SpyTag or SpyCatcher was successfully fused to the C-terminus of Dead streptavidin subunits. We were able to generate chimeric tetramers with n (0 ≤ n ≤ 4) biotin binding sites and 4-n SpyTag or SpyCatcher binding sites. Chimeric SpyAvidin tetramers bound precise numbers of ligands fused to biotin or SpyTag/SpyCatcher. Mixing chimeric tetramers enabled assembly of SpyAvidin octamers (8 subunits) or eicosamers (20 subunits). We validated assemblies using electrophoresis and native mass spectrometry. Eicosameric SpyAvidin was used to cluster trimeric major histocompatibility complex (MHC) class I:β2-microglobulin:peptide complexes, generating an assembly with up to 56 components. MHC eicosamers surpassed the conventional MHC tetramers in acting as a powerful stimulus to T cell signaling. Combining ultrastable noncovalent with irreversible covalent interaction, SpyAvidins enable a simple route to create robust nanoarchitectures.

Comparative studies on steam gasification of ash-free coals and their original raw coals

Catalytic gasification of raw coals at mild condition is not realized yet mainly due to deactivation of catalysts via their irreversible interaction with mineral matters in coal. As a means to achieve repeated use of catalysts, four different ash-free coals (AFCs) containing less than 0.2 wt% ash are produced in this work. Steam gasification of ash-free coals (AFCs) and their parent raw coals of various ranks ranging from lignite (Eco) to coking coal (Posco) is performed in a fixed bed reactor at 700–900 °C. Regardless of the rank of the parent raw coals, all the AFCs behave like a highly carbonized coal such that their gasification rate are similarly slow and they exhibit relatively low H2/CO ratio. The steam gasification and associated CO to CO2 conversion of the AFCs are, however, significantly enhanced by K2CO3, resulting in the higher H2/CO and CO2/CO molar ratio.

Comparison Of Plasma Kallikrein Inhibition By The Endogenous C1-Inhibitor Versus DX-2930, a Monoclonal Antibody Inhibitor

Dysregulated plasma kallikrein proteolytic activity leads to edematous attacks in hereditary angioedema (HAE) and has been associated with inflammation and thrombosis. Plasma kallikrein (pKal) is a serine protease that circulates as prekallikrein, a zymogen, which, together with factor XII (FXII) and high molecular weight kininogen (HMWK), constitutes the contact system. Activation of the contact system following assembly of FXII, HMWK, and prekallikrein on a negatively charged surface promotes inflammation via the generation of bradykinin and triggers intrinsic pathway coagulation via formation of activated coagulation factor XIa. Normal hemostasis appears not to be mediated by the contact system as individuals deficient in contact system proteins are not at risk for bleeding. However, the contact system has been shown to be pathologically activated by agents that include misfolded proteins, platelet polyphosphate, and implanted devices. Therefore, pharmacologic modulation of the contact system may attenuate thrombosis and inflammation without disrupting normal hemostasis. C1 inhibitor (C1-INH) is a serpin and a key endogenous, protein-based, inhibitor of pKal activity. HAE is caused by autosomal dominant mutations in the C1-INH gene resulting in functional protein levels that are approximately 30% or less than normal (16-33 mg/dL or 1.6-3.3 μM). Prekallikrein is present in plasma at a concentration of approximately 500 nM and it has been estimated that only 30-110 nM is converted to active pKal during an HAE attack. This study investigates the requirement for super-stoichiometric amounts of endogenous C1-INH to adequately regulate pKal activity. In vitro enzyme inhibition kinetics experiments with purified proteins show that the need for high concentrations of C1-INH is likely due to its relatively slow association rate constant (1.7 x 104 M-1s-1). In contrast, DX-2930, a human monoclonal antibody inhibitor of pKal being developed for prophylactic treatment of HAE, potently inhibited pKal (Ki = 125 pM) with a faster association rate constant (3.4 x 106 M-1s-1). Contact activation was observed in human plasma activated by the addition of ellagic acid and monitored using a pKal-selective synthetic peptide substrate. Consistent with the data obtained using purified proteins, the apparent IC50 observed upon adding exogenous C1-INH to normal human plasma was approximately 100-fold higher than that of DX-2930. Using a Western blot assay to monitor endogenous HMWK cleavage in activated plasma we similarly observed that stoichiometric additions of DX-2930 were sufficient to prevent HMWK proteolysis by active pKal; whereas significantly higher concentrations of C1-INH (e.g. 1 µM) were required to block HMWK proteolysis. Active pKal can bind endothelial cells via interactions between the non-catalytic domain of pKal with HMWK, which binds receptors (urokinase receptor, cytokeratin 1, and the globular C1q receptor) present on endothelial cells. Cell bound pKal is likely to be a physiologically relevant form of the enzyme and may provide an explanation for attack localization in HAE. In this study, active pKal was assembled in vitro on cultured human umbilical vein endothelial cells (HUVEC) and binding of a range of concentrations of either biotinylated C1-INH or biotinylated DX-2930 was observed using streptavidin-horseradish peroxidase as detection. The data obtained demonstrates that C1-INH bound cell-associated pKal with > 200-fold less potency than DX-2930. Regarding protease inhibition specificity, while DX-2930 did not inhibit any of 20 tested serine proteases at a concentration of 1 µM, C1-INH is known to inhibit multiple serine proteases. This study demonstrates that effective regulation of pKal activity requires high concentrations of C1-INH (≥ 1 µM), which are necessary to drive the kinetics of this second order, irreversible interaction. These high inhibitory concentrations of C1-INH match the normal range and provide a potential kinetic mechanism for why HAE attacks can occur at C1-INH levels that exceed expected levels of activated pKal. Furthermore, the broad specificity of C1-INH towards other proteases that could be activated during disease could sufficiently deplete C1-INH levels and thereby lead to dysregulated pKal activity. Disclosures:Sexton:Dyax Corp: Employment. Kenniston:Dyax Corp: Employment. Faucette:Dyax Corp: Employment. Nixon:Dyax Corp: Employment. TenHoor:Dyax Corp: Employment. Chyung:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Design, synthesis and evaluation of a cellular stable and detectable biotinylated fumagillin probe and investigation of cell permeability of fumagillin and its analogs to endothelial and cancer cells

Fumagillin (1), a natural product of fungal origin, and its analogs were discovered to be extremely potent and highly selective inhibitors restraining endothelial cell proliferation in vitro by covalently binding to MetAP2. In order to further understand the unclear biological mechanisms and pharmacological processes of fumagillin and its derivatives, fumagillin–biotin conjugate 8 was designed and synthesized, which is linked with a 27-atom connection chain and by urethane (carbamate) bonds between fumagillol and d-norbiotinamine. The conjugate 8 shows comparable activity and selectivity against HUVEC proliferation as fumagillin. It was demonstrated that the conjugate 8 is stable inside the cell and its linker is of a suitable length for the detection of biotin in native and denatured conditions. Using the conjugate 8, it was determined that the cell permeability of fumagillin (1) and its analogs are not responsible for their inhibitory activity difference against the proliferation of endothelial and cancer cells. Furthermore, we confidently believe that our present strategy is a versatile and convenient method for investigating drug's cell permeability along with other studies regardless of reversible or irreversible interaction between the drug and binding target/s.

Hydrogen production via steam gasification of ash free coals

The kinetics of the coal to hydrogen conversion can be significantly enhanced by introducing catalysts. The catalysts are, however, commonly deactivated by irreversible interaction with mineral matters in coal. This work addresses hydrogen production via steam gasification of ash free coals. Following the production of ash free coals (AFCs) derived from various raw coals (brown, bituminous, and coking coal), fixed-bed steam gasification of the AFCs was performed as a function of temperature and which was compared with one another and also with that of the matching raw coals. In the absence of a catalyst, AFCs produced from different parent coals exhibited similarly low gasification reactivity, comparable to a high rank coal (coking coal) at 700 °C. As expected, the reaction became faster with increasing temperature in the range, 700–900 °C. The steam gasification of AFCs was highly activated by K2CO3 above 700 °C. It was very likely that water–gas shift reaction associated with the gasification of AFCs was also catalyzed.

Disposable Attenuated Total Reflection-Infrared Crystals from Silicon Wafer: A Versatile Approach to Surface Infrared Spectroscopy

Attenuated total reflection-infrared (ATR-IR) spectroscopy is increasingly used to characterize solids and liquids as well as (catalytic) chemical conversion. Here we demonstrate that a piece of silicon wafer cut by a dicing machine or cleaved manually can be used as disposable internal reflection element (IRE) without the need for polishing and laborious edge preparation. Technical aspects, fundamental differences, and pros and cons of these novel disposable IREs and commercial IREs are discussed. The use of a crystal (the Si wafer) in a disposable manner enables simultaneous preparation and analysis of substrates and application of ATR spectroscopy in high temperature processes that may lead to irreversible interaction between the crystal and the substrate. As representative application examples, the disposable IREs were used to study high temperature thermal decomposition and chemical changes of polyvinyl alcohol (PVA) in a titania (TiO(2)) matrix and assemblies of 65-450 nm thick polystyrene (PS) films.